Search PubMed⌕ Search

Biomedical subjects

G Medoff

Publications and source records attributed to G Medoff.

At least 55 records · Page 3Linked to original sources

Heat shock 70 gene is differentially expressed in Histoplasma capsulatum strains with different levels of thermotolerance and pathogenicity.

The response to heat shock has been examined in two strains of the dimorphic pathogenic fungus Histoplasma capsulatum, which differ considerably in thermotolerance and pathogenicity. The gene for the 70 kD heat shock protein (hsp70) was isolated using a Drosophila hsp70 gene to screen a cosmid library of the DNA from the temperature-sensitive Downs strain (low level of thermotolerance for mice). Using the cloned gene as a probe, we have measured the transcription of the endogenous hsp70 gene at 25 degrees C and in response to temperature shift to 34 degrees, 37 degrees and 40 degrees C, temperatures that trigger the mycelial to yeast phase transition in this fungus. The gene is constitutively transcribed at low levels, both in the yeast and the mycelial stages. Synthesis of hsp70 mRNA was transiently increased 1 to 3 h after the temperature shifts. By Northern analysis, peak levels of transcription were shown to occur at 34 degrees C in the Downs strain and at 37 degrees C in the more pathogenic G222B strain. Our results are consistent with reports in which it has been shown that heat shock gene expression is part of temperature adaptation and probably developmental processes. The low levels of transcription of the hsp70 gene in the Downs strain at 37 degrees C correlate with its greater temperature sensitivity and low level of virulence.

Animals↗

Morphogenesis and pathogenicity of Histoplasma capsulatum.

The sulfhydryl blocking agent p-chloromercuriphenylsulfonic acid (PCMS) irreversibly inhibited the mycelium-to-yeast transitions of two virulent strains of Histoplasma capsulatum, G184A and G222B, when the temperature of incubation was raised to 37 degrees C, and the block persisted even after the cultures were washed free of PCMS. Instead of transforming to yeast cells, PCMS-treated mycelia continued to grow as mycelia at the elevated temperatures. A less virulent strain (Downs) was more temperature sensitive, but it showed a similar irreversible effect at 34 degrees C. Therefore, the mycelium-to-yeast transition of H. capsulatum is not required for the adaptation of mycelia to elevated temperatures but probably results from the temperature-dependent activation of yeast-specific genes. The transition to yeast is inferred to be obligate for pathogenicity in mice because PCMS-treated mycelia failed to cause infection, and no fungi were seen in tissues after PCMS-treated mycelia were injected into mice.

4-Chloromercuribenzenesulfonate↗

Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis.

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.

Adenosine Triphosphate↗

Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum.

p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens.

4-Chloromercuribenzenesulfonate↗

Amphotericin B-induced oxidative damage and killing of Candida albicans.

Amphotericin B (AmB) is known to bind to ergosterol in fungal cell membranes, but the precise mechanism of its toxicity to cells is as yet poorly understood. AmB autooxidizes, and it is possible that its antifungal effects could result from oxidative damage. Exposure of protoplasts of Candida albicans to AmB under hypoxic conditions reduced protoplast lysis by as much as 80% compared with incubations in air. Protoplasts were protected from AmB-induced lysis by exogenous catalase and/or superoxide dismutase (SOD). Whole cells of C. albicans were protected by exogenous catalase from AmB-induced leakage of [3H]leucine and from killing by AmB. Cells grown on medium inducing high levels of endogenous catalase were resistant to AmB-induced growth inhibition. In contrast, AmB-induced K+ leakage was not hindered under hypoxic conditions or in the presence of catalase or SOD. Thus the lethal and lytic effects of AmB on C. albicans cells and protoplasts, but not prelethal AmB-induced K+ leakage, are mediated by oxidative damage.

Amphotericin B↗

Comparison of the in vitro and in vivo activity of the bis-triazole derivative UK 49,858 with that of amphotericin B against Histoplasma capsulatum.

The antifungal activity of UK 49,858, a difluorophenyl bis-triazole derivative, was evaluated in vitro against seven strains of Histoplasma capsulatum and in vivo in AKR and C57BL/6 murine models of histoplasmosis. UK 49,858 had a lower toxicity for AKR and C57BL/6 mice than amphotericin B did. The therapeutic index of UK 49,858 was 4.3 for AKR mice and 7.1 for C57BL/6; with amphotericin B it was 2 for both mouse strains. Given orally, UK 49,858 compared favorably with amphotericin B given intraperitoneally in either AKR or C57BL/6 mice infected with H. capsulatum.

Amphotericin B↗

Sensitivity of Candida albicans to amphotericin B administered as single or fractionated doses.

Candida albicans cells were exposed to equal concentrations of amphotericin B (AmB) administered either as a single large dose or as repeated small doses. Toxicity to C. albicans cells was less pronounced when AmB was administered in fractionated doses. Increased catalase activity in C. albicans cells was induced after exposure to fractionated doses of AmB; this increase may have contributed to the greater resistance of cells to AmB used according to this schedule.

Amphotericin B↗

Toxicity and induction of resistance to Listeria monocytogenes infection by amphotericin B in inbred strains of mice.

Amphotericin B (AmB) treatment before infection with the bacterium Listeria monocytogenes prolonged survival of AKR mice but shortened survival of C57BL/6 mice compared with survival of untreated infected controls. C57BL/6 mice were also more sensitive to the acute toxic effects of AmB than AKR mice, as were (C57BL/6 X AKR)F1 hybrid mice. Spleen cells and erythrocytes (RBCs) from the C57BL/6 and the F1 hybrid mice were both more sensitive to the lytic and lethal effects of AmB than corresponding cells from AKR mice. Biochemical analysis indicated that catalase levels in RBCs from C57BL/6 and F1 hybrid mice were about 60% of those found in RBCs from AKR mice. The lysis by AmB of RBCs from all these strains of mice was inhibited by catalase or incubation in a low-oxygen environment. These findings suggest that (i) the low catalase levels in C57BL/6 and F1 hybrid mice may limit the protection of cells from the oxidant damage involved in AmB action, and (ii) the toxicity which occurs at low concentrations of AmB in the mouse strains with low intracellular catalase levels may interfere with or ablate the AmB-induced increases in mouse resistance to L. monocytogenes infection.

Amphotericin B↗

Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms.

Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1.

Africa↗

Correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum.

We compared the mycelial to yeast transitions of the Downs strain of Histoplasma capsulatum (low level of virulence) with those of G184A and G222B, two more virulent strains having different levels of pathogenicity for mice. When the morphological transitions are initiated by a temperature shift from 25 degrees to 37 degrees C, all three strains undergo similar physiological changes, but these are less severe in G184A and G222B than in the Downs strain. The transitions from mycelial to yeast morphology in both of the more virulent strains are also one-third more rapid than in Downs. We also find that the differences in temperature sensitivity of the three strains can be correlated with the temperature required for complete uncoupling of oxidative phosphorylation. The differences in sensitivity to elevated temperatures extend to the growth of yeast cells of all three strains. Considered together, our results suggest that sensitivity to elevated temperatures may be a key factor accounting for differences in virulence and that uncoupling of oxidative phosphorylation may be the primary event in the morphological transition in all three strains.

Adenosine Triphosphate↗

Two mechanisms of synergism when amphotericin B is used in combination with actinomycin D or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea against the human promyelocytic leukemia cell line HL-60.

The toxic effects of the combinations of amphotericin B (AmB) and actinomycin D or AmB and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were measured against the human promyelocytic leukemia cells HL-60. The toxicities of both drug combinations were greater than the additive toxicity of each of the drugs used singly, but the exact conditions under which synergism was achieved differed for each combination. The synergism achieved by the AmB-actinomycin combination was accompanied by an AmB-induced increase in uptake of actinomycin D by the HL-60 cells, whereas the synergism of the AmB-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea combination could be linked to potentiation by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea of AmB-induced oxidative injury. These results indicate that the synergism of these two drug combinations was caused by different mechanisms.

Amphotericin B↗

Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B.

Lysis of human erythrocytes induced by amphotericin B was retarded when the oxygen tension of the incubation mixture was reduced or when the antioxidant catalase was added; lysis was accelerated when cells were preincubated with the prooxidant ascorbate. In the atmosphere of reduced oxygen tension, the erythrocytes containing carboxyhemoglobin lysed at a slower rate than did the cells containing oxyhemoglobin. Consistent with a role for oxidative damage in lysis, the mixture of erythrocytes and amphotericin B showed an increase in malonyldialdehyde, the product of peroxidation, which paralleled the progression of hemolysis. In contrast, the permeabilizing effect of amphotericin B, measured as a decrease in intracellular K+, was not affected by changes in oxygen tension, catalase, or ascorbate treatment. These results imply that oxidant damage is involved in the lytic, but not in the permeabilizing, action of amphotericin B.

Amphotericin B↗

In vitro and in vivo comparisons of amphotericin B and N-D-ornithyl amphotericin B methyl ester.

N-D-Ornithyl amphotericin B methyl ester (N-D-ornithyl AmE) has a lower toxicity for animals than does amphotericin B (AmB), and peak serum levels can be achieved that are fourfold higher than those obtained with an equivalent dose of AmB. However, N-D-ornithyl AmE has one-fourth the in vitro activity and between one-fifth and one-eighth the in vivo activity of AmB. N-D-ornithyl AmE and the corresponding lysyl derivative also lack the immunoadjuvant effects of AmB.

Adjuvants, Immunologic↗

Cross-reacting human and rabbit antibodies to antigens of Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae.

Using Western blots of electrophoretically separated antigens, we show that human antibodies react most frequently to antigens shared by three fungi (Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae). Reactivity to antigens specific for individual fungi was relatively uncommon. The pattern of reactivity could not distinguish infected patients from uninfected controls. Rabbits immunized with extracts of each fungus also produced antibodies to cross-reactive or shared antigens of the other two fungi. Furthermore, preimmune sera showed similar but lower reactivity with the same fungal antigens. We believe that the preimmunization antibodies, which probably resulted from earlier fungal colonization or inapparent infections, predisposed the immune responses elicited by the vaccinations. A similar mechanism likely explains the results with human sera.

Animals↗

Growth inhibition of Cryptococcus neoformans by cloned cultured murine macrophages.

Cloned and unselected bone marrow-derived macrophage cell lines were obtained from A/J, AKR/J, BIO.A(5R), CBA/J, DBA/2, HPC, NZW, and [NZB X NZW]F1 mice, and their interactions were studied in vitro with a lightly encapsulated natural serotype A isolate of Cryptococcus neoformans. Growth inhibition of C. neoformans was seen with all of the cell lines, as determined by enumeration of colony-forming units. Inhibition was enhanced by a high concentration (8%) of fresh mouse serum and was the same for serum obtained from AKR/J (C5 deficient) and BIO.A (C5 normal) mice. Macrophage incubation with fresh AKR/J serum which had been absorbed with heat-killed Cryptococcus cells also inhibited C. neoformans growth. Heat-inactivation, EDTA addition or anti-C3 antibody treatment of fresh serum abolished the opsonic activity for C. neoformans, while EGTA addition to fresh serum was without effect on opsonization. In addition, neither IgM nor IgG1 murine monoclonal antibodies specific for C. neoformans enhanced phagocytosis or killing of the yeast by macrophages. These findings are consistent with the interpretation that C3b is an important modulator of interactions between macrophages and C. neoformans.

Animals↗